Receptor-mediated endocytosis is critical for sustaining and regulating cell proliferation. Receptor-mediated endocytosis of transferrin (Tfn), for example, provides an essential nutrient for cell growth. Antibodies which inhibit Tfn-R internalization also inhibit tumor growth and metastasis. Internalization of tyrosine-kinase containing growth factor receptors, such as the epidermal growth factor receptor (EGF-R) is not required for ligand-induced mitogenesis but rather to attenuate the cellular response to EGF. Failure to regulate the signal strength and consequential cellular response to EGF by endocytosis leads to cell transformation. The importance of these processes to normal and transformed cell growth is clear. Our long-term objectives are to understand the molecular mechanisms which govern receptor-mediated endocytosis in general and the down regulation of mitogenic receptors, specifically. Towards this goal, we have developed a novel cell-free system for receptor-mediated endocytosis of Tfn and EGF. Extensive biochemical and morphological characterization of this system has established its usefulness as a functional assay for the mechanistic dissection of receptor-medicated endocytosis. Our recent results using this in vitro system have revealed several distinct biochemical requirements for endocytosis of EGF-R which mirror our expectations from in vivo studies. Based on these results we propose to characterize the biochemical requirements which distinguish the ligand- induced internalization of EGF-R from the constitutive internalization of Tfn-R, and to identify the molecular basis for these differences. In so doing we will identify factors which constitute the endocytic apparatus specifically required for EGF-R internalization. They are complementary to and will be performed in close collaboration with Gordon Gill (Project 4) whose molecular genetic dissection of the EGF-R serves to identify the structural features of the receptor specifically recognized by the endocytic apparatus. Our results should provide important new insight into the regulation of the cellular response to EGF.